Conjugation reactions

ABSTRACT

An initiator for the terminal group of the polymer product of an atom or gruop radical transfer polymerisation has an activated carboxyl or an amine group which is reacted with an amine or carboxyl (respectively) group containing biologically active compound. The initiator is preferably 4-(3-(2-bromo, 2-methyl-propionate)phenyl)-propionic acid N-hydroxysuccinimide ester or 2-bromo, 2-methyl-propionic acid N-hydroxysuccinimide ester. The monomers preferably comprise a zwitterionic monomer such as 2-methacryloxyethyl-2′-trimethyl ammoniumethyl phosphate inner salt.

The present invention relates to forming conjugates of polymers andbiologically active compounds, and to functional polymer precursorstherefore.

We have described in WO-A-2003062290 (unpublished at the priority datehereof) the concept of taking a biologically active drug molecule andreacting it with a compund so as to functionalise it with an appropriatebrominated moiety. The modified drug is capable of initiating an atomtransfer radical polymerisation (ATRP) with monomers such as2-methacryloyloxyethyl-2′-trimethylammonium ethyl phosphate inner salt.The compound 2-bromo-2-methyl propionic acid bromide is one suchbromine-functionalising agent, the acid bromide portion reacting withany active hydroxyl group on the drug compound to produce a brominatedanalogue of a methacrylate moiety that has been shown to be eminentlysuitable for initiating ATRP (see works of Matyjaszewski, Armes andHaddleton). With this functionalising agent, this reaction is onlypossible in cases where the drug molecule and the 2-bromo, 2-methylpropionic acid bromide are both soluble in some organic solvent that isnot reactive towards the acid bromide. In that specification we alsodescribe a bromine-functionalising agent which is used to acylate anamine group, namely of a protein. The subject matter has a commonpriority date as claims of the present case and of example 4 herein.

It may be desirable to functionalise large biological entities such asproteins and antibodies with polymers. Roche and Schering-Plough bothhave on the market successful interferon-based products that have beenmodified by PEGylation of the biological entity. The attachment ofpolyethylene glycol chains to interferon has improved its plasmahalf-life (i.e. reduced the rate at which the body removes the antibodyfrom the bloodstream), essentially by reduced renal clearance andlowering opsonisation (protein binding). It is of interest to modifysuch biological entities with other polymers, particularly those basedon phosphorylcholine because of their enhanced haemocompatibility.

In U.S. Pat. No. 6,310,149 ATRP processes are described in which theinitiator comprises a functional group which is a derivative of anorganic acid. The acid may be a carboxylic acid or alternatively aphosphorus based acid or sulphonic acid. Examples of initiators areesters of various acids. Monomers which are polymerised includemethylmethacrylate, styrene, benzylmethacrylate and2-hydroxyethylmethacrylate. The polymerisations appear to besolvent-free, that is liquid monomer is the liquid medium for thepolymerisation reaction.

Wang, J-S, et al in Polym. Mater. Sci. Eng. 73 (1995) 416 to 417describe the use of bis-functional initiators for ATRP, whereby theresidual group derived from the initiator may be used in subsequentreactions. Examples of end groups or precursors thereof are carboxylgroups, hydroxyl groups and cyano groups.

The present invention relates to a new process for forming abiologically active conjugate compound comprising a conjugation step inwhich a biologically active starting material of the general formula I

is reacted in an amide bond forming step with a reagent of the generalformula II

to form an amide-linked conjugate,

in which R is a biologically active moiety;

L¹ is a bond or a divalent organic linker; and

X¹ is —NHR⁶ or —COR⁷, in which R⁶ is hydrogen, C₁₋₆ alkyl, aryl or anamine activating group and R⁷ is hydroxyl or a carboxyl activatinggroup;

X² is selected from the group consisting of a polymer formed fromethylenically unsaturated monomers and joined through a terminal group,Cl, Br, I, OR¹⁰, SR¹⁴, SeR¹⁴, OP(═O)R¹⁴, OP(═O)(OR¹⁴)₂, O—N(R¹⁴)₂ andS—C(═S)N(R¹⁴)₂, where R¹⁰ is alkyl of from 1 to 20 carbon atoms in whicheach of the hydrogen atoms may be independently replaced by halide, R¹⁴is aryl or a straight or branched C₁-C₂₀ alkyl group, and where anN(R¹⁴)₂ group is present, the two R¹⁴ groups may be joined to form a 5-or 6-membered heterocyclic ring;

R¹ and R² are each independently selected from the group consisting ofH, halogen, C₁-C₂₀ alkyl, C₃-C₈ cycloalkyl, C(═O)R¹⁵, C(═O)NR¹⁶R¹⁷,COCl, OH, CN, C₂-C₂₀ alkenyl, oxiranyl, glycidyl, aryl, heterocyclyl,aralkyl and aralkenyl, in any of which the alkyl, alkenyl or aryl,heterocyclyl or cycloalkyl groups there may be from 1 to 3 substituentsselected from the group consisting of hydrogen, hydroxy C₁-C₄ alkoxy,acyloxy, aryl, heterocyclyl, C(═O)R¹⁵, C(═O)NR¹⁶R¹⁷, oxyranyl andglycidyl; R¹⁵ is alkyl of from 1 to 20 carbon atoms, alkoxy of from 1 to20 carbon atoms, oligo(alkoxy) in which each alkoxy group has 1 to 3carbon atoms, aryloxy or heterocyclyloxy any of which groups may havesubstituents selected from optionally substituted alkoxy, oligoalkoxy,amino (including mono- and di-alkyl amino and trialkyl ammonium, whichalkyl groups, in turn may have substituents selected from acyl, acyloxy,alkoxy, alkoxycarbonyl, alkenoxycarbonyl, aryl and hydroxy), andhydroxyl groups;

R¹⁶ and R¹⁷ are independently H or alkyl of from 1 to 20 carbon atomswhich alkyl groups, in turn may have substituents selected from alkoxy,acyl, acyloxy, alkoxycarbonyl, alkenoxycarbonyl, aryl and hydroxy, orR¹⁶ and R¹⁷ may be joined together to form an alkanediyl group of from 2to 5 carbon atoms, thus forming a 3- to 6-membered ring.

R³ and R⁴ are independently selected from hydrogen, and C₁₋₆ alkyl or R³and R⁴together are ═O or ═NR⁸ where R⁸ is hydrogen or C₁₋₄ alkyl;

R⁵ is a bond, —O—, —S— or a divalent organic group;

L² is a bond or a divalent linker and,

where X¹ is NHR⁶, X³ is COR⁷ and

where X¹ is COR⁷, X³ is NHR⁶ and

r is an integer of at least 1.

In the conjugation reaction an amide bond is formed from the reaction ofthe carboxyl functionality COR⁷ and the amine functionality —NHR⁶, of X¹and X³. Known activating agents may be used to assist the formation ofthe amide bond, such as carbodiimides and/or N-hydroxysuccinimide.Preferably R⁷ is an N-succinimidyloxy group. R⁶ may be a C₁₋₆ alkyl oraryl group, but is preferably hydrogen.

Preferably the conjugation reacting step is carried out in a proticsolvent e.g. a lower alkanol and/or, most preferably, water.

Where X² is other than a polymer chain, the amide conjugate product maybe used as an initiator for an atom- or group transfer radicalpolymerisation reaction.

The ethylenically unsaturated monomers used in such a polymerisation maybe any which may be codissolved with the amide in a suitable solvent forsuch a polymerisation, such as an organic or aqueous solvent. Theinvention is of most utility for polymerising hydrophilic monomers, thatis which are soluble in water, lower alkanols (C₁₋₄-alkanols, includingglycols) or glycol ethers. The polymerisation is generally conducted inthe presence of one of these solvents, in which the monomers aredissolved. Conveniently the solvent includes water.

Suitable monomers have the general formula III

in which R²³ is selected from the group consisting of hydrogen, halogen,C₁₋₄ alkyl and groups COOR²⁷ in which R²⁷ is hydrogen and C₁₋₄ alkyl;

R²⁴ is selected from the group consisting of hydrogen, halogen and C₁₋₄alkyl;

R²⁵ is selected from the group consisting of hydrogen, halogen, C₁₋₄alkyl and groups COOR²⁷ provided that R²³ and R²⁵ are not both COOR²⁷;and

R²⁶ is selected from the group consisting of C₁₋₁₀ alkyl, C₁₋₂₀alkoxycarbonyl, mono- and di-(C₁₋₂₀ alkyl) amino carbonyl, C₆₋₂₀ aryl(including alkaryl), C₇₋₂₀ aralkyl, C₆₋₂₀ aryloxycarbonyl,C₇₋₂₀-aralkyloxycarbonyl, C₆₋₂₀ arylamino carbonyl, C₇₋₂₀ aralkyl-aminocarbonyl, hydroxyl and carboxylic C₂₋₁₀ acyloxy groups, any of which mayhave one or more substituents selected from the group consisting ofhalogen atoms, alkoxy, oligo-alkoxy, aryloxy, acyloxy, acylamino, amine(including mono and di-alkyl amino and trialkylammonium in which thealkyl groups may be substituted), carboxyl, sulphonyl, phosphoryl,phosphino, (including mono- and di-alkyl phosphine andtri-alkylphosphonium), zwitterionic, hydroxyl, vinyloxycarbonyl andother vinylic and allylic groups, and reactive silyl and silyloxygroups, such as trialkoxysilyl groups;

or R²⁶ and R²⁵ or R²⁵ and R²³ may together form —CONR²⁸CO in which R²⁸is a C₁₋₂₀ alkyl group.

It is preferred for at least two of the groups R²³, R²⁴, R²⁵ and R²⁶ tobe halogen or, more preferably, hydrogen atoms. Preferably R²³ and R²⁴are both hydrogen atoms. It is particularly preferred that compound ofgeneral formula III be a styrene-based or (alk) acrylic based compound.In styrene based compounds R²⁶ represents an aryl group, especially asubstituted aryl group in which the substituent is an amino alkyl group,a carboxylate or a sulphonate group. Where the comonomer is an (alk)acrylic type compound, R²⁶ is an alkoxycarbonyl, an alkyl aminocarbonyl, or an aryloxy carbonyl group R²³ and R²⁴ are each hydrogen andR²⁵ is hydrogen or C₁₋₄ alkyl. Most preferably in such compounds R²⁶ isa C₁₋₂₀ -alkoxy carbonyl group, optionally having a hydroxy substituent.(Alk) acrylic compounds are generally methacrylic in which case R²⁵ ismethyl.

Preferably the monomers include a zwitterionic monomer having thegeneral formula IVYBX   IVin which Y is an ethylenically unsaturated group selected fromH₂C═CR¹⁷—CO-A-, H₂C═CR¹⁷—C₆H₄-A¹-, H₂C═CR¹⁷—CH₂A¹⁸,R²O—CO—CR¹⁷═CR¹⁷—CO—O, R¹⁷CH═CH—CO—O—, R¹⁷CH═C(COOR¹⁸)CH₂—CO—O,

A is —O— or NR¹⁹;

A¹ is selected from a bond, (CH₂)_(l)A² and (CH₂)_(l)SO₃— in which l is1 to 12;

A² is selected from a bond, —O—, O—CO—, CO—O, CO—NR¹—, —NR¹—CO,O—CO—NR¹—, NR¹—CO—O—;

R¹⁷ is hydrogen or C₁₋₄ alkyl;

R¹⁹ is hydrogen, C₁₋₄₋ alkyl or BX;

R¹⁸ is hydrogen or C₁₋₄ alkyl;

B is a bond, or a straight branched alkanediyl, alkylene oxaalkylene, oralkylene (oligooxalkylene) group, optionally containing one or morefluorine substituents; and

X is a zwitterionic group.

Preferably X is an ammonium, phosphonium, or sulphonium phosphate orphosphonate ester zwitterionic group, more preferably a group of thegeneral formula V

in which the moieties A³ and A⁴, which are the same or different, are—O—, —S—, —NH— or a valence bond, preferably —O—, and W⁺ is a groupcomprising an ammonium, phosphonium or sulphonium cationic group and agroup linking the anionic and cationic moieties which is preferably aC₁₋₁₂-alkanediyl group,

preferably in which W⁺ is a group of formula —W¹—N⁺R²⁰ ₃, —W¹—P⁺R²¹ ₃,—W¹—S⁺R²¹ ₂ or —W¹-Het⁺ in which:

W¹ is alkanediyl of 1 or more, preferably 2-6 carbon atoms optionallycontaining one or more ethylenically unsaturated double or triple bonds,disubstituted-aryl (arylene), alkylene arylene, arylene alkylene, oralkylene aryl alkylene, cycloalkanediyl, alkylene cycloalkyl, cycloalkylalkylene or alkylene cycloalkyl alkylene, which group W¹ optionallycontains one or more fluorine substituents and/or one or more functionalgroups; and

either the groups R²⁰ are the same or different and each is hydrogen oralkyl of 1 to 4 carbon atoms, preferably methyl, or aryl, such asphenyl, or two of the groups R²⁰ together with the nitrogen atom towhich they are attached form an aliphatic heterocyclic ring containingfrom 5 to 7 atoms, or the three groups R²⁰ together with the nitrogenatom to which they are attached as heteroaromatic ring having 5 to 7atoms, either of which rings may be fused with another saturated orunsaturated ring to form a fused ring structure containing from 5 to 7atoms in each ring, and optionally one or more of the groups R²⁰ issubstituted by a hydrophilic functional group, and

the groups R²¹ are the same or different and each is R²⁰ or a groupOR²⁰, where R²⁰ is as defined above; or

Het is an aromatic nitrogen-, phosphorus- or sulphur-, preferablynitrogen-, containing ring, for example pyridine.

Monomers in which X is of the general formula in which W⁺ is W¹N^(Γ)R²⁰₃ may be made as described in our earlier specification WO-A-9301221.Phosphonium and sulphonium analogues are described in WO-A-9520407 andWO-A-9416749.

Generally a group of the formula V has the preferred general formula VI

where the groups R²² are the same or different and each is hydrogen orC₁₋₄ alkyl, and m is from 1 to 4, in which preferably the groups R²² arethe same preferably methyl.

In phosphobetaine based groups, X may have the general formula VII

in which A⁵ is a valence bond, —O—, —S— or —NH—, preferably —O—;

R⁹ is a valence bond (together with A⁵) or alkanediyl, —C(O)alkylene- or—C(O)NH alkylene preferably alkanediyl, and preferably containing from 1to 6 carbon atoms in the alkanediyl chain;

W² is S, PR³⁰ or NR³⁰;

the or each group R³⁰ is hydrogen or alkyl of 1 to 4 carbon atoms or thetwo groups R³⁰ together with the heteroatom to which they are attachedform a heterocyclic ring of 5 to 7 atoms;

R³¹ is alkanediyl of 1 to 20, preferably 1 to 10, more preferably 1 to 6carbon atoms;

A⁶ is a bond, NH, S or O, preferably O; and

R³² is a hydroxyl, C₁₋₁₂ alkyl, C₁₋₁₂ alkoxy, C₇₋₁₈ aralkyl, C₇₋₁₈-aralkoxy, C₆₋₁₈ aryl or C₆₋₁₈ aryloxy group.

Monomers comprising a group of the general formula VII may be made bymethods as described in JP-B-03-031718, in which an amino substitutedmonomer is reacted with a phospholane.

In compounds comprising a group of the general formula VII, it ispreferred that

A⁵ is a bond;

R²⁹ is a C₂₋₆ alkanediyl;

W² is NR⁷:

each R⁷ is C₁₋₄ alkyl;

R³¹ is C₂₋₆ alkanediyl;

A⁶ is O; and

R³² is C₁₋₄ alkoxy.

Alternatively X may be a zwitterion in which the anion comprises asulphate, sulphonate or carboxylate group.

One example of such a group is a sulphobetaine group, of the generalformula VIII

where the groups R³³ are the same or different and each is hydrogen orC₁₋₄ alkyl and s is from 2 to 4.

Preferably the groups R³³ are the same. It is also preferable that atleast one of the groups R³³ is methyl, and more preferable that thegroups R³³ are both methyl.

Preferably s is 2 or 3, more preferably 3.

Another example of a zwitterionic group having a carboxylate group is anamino acid moiety in which the alpha carbon atom (to which an aminegroup and the carboxylic acid group are attached) is joined through alinker group to the backbone of the biocompatible polymer. Such groupsmay be represented by the general formula IX

in which A⁷ is a valence bond, —O—, —S— or —NH—, preferably —O—,

R³⁴ is a valence bond (optionally together with A⁷) or alkanediyl,—C(O)alkylene- or —C(O)NHalkylene, preferably alkanediyl and preferablycontaining from 1 to 6 carbon atoms; and

the groups R³⁵ are the same or different and each is hydrogen or alkylof 1 to 4 carbon atoms, preferably methyl, or two or three of the groupsR³⁵, together with the nitrogen to which they are attached, form aheterocyclic ring of from 5 to 7 atoms, or the three group R³⁵ togetherwith the nitrogen atom to which they are attached form a fused ringheterocyclic structure containing from 5 to 7 atoms in each ring.

Another example of a zwitterion having a carboxylate group is a carboxybetaine —N^(Γ)(R³⁶)₂(CH₂)_(u)COO^(Γ) in which the R³⁶ groups are thesame or different and each is hydrogen or C₁₋₄ alkyl and u is 2 to 6,preferably 2 or 3.

In the zwitterionic monomer of the general formula IV it is preferredthat the ethylenic unsaturated group Y is H₂C═CR¹⁷—CO-A-. Such (alk)acrylic moieties are preferably methacrylic, that is in which R¹⁷ ismethyl, or acrylic, in which R¹⁷ is hydrogen. Whilst the compounds maybe (alk)acrylamido compounds, that is in which A is NR¹⁹, in which caseR¹⁹ is preferably hydrogen, or less preferably, methyl, most preferablythe compounds are esters, that is in which A is O.

In monomers of the general formula IV, especially where Y is thepreferred (alk)acrylic group, B is most preferably an alkanediyl group.Whilst some of the hydrogen atoms of such group may be substituted byfluorine atoms, preferably B is an unsubstituted alkanediyl group, mostpreferably a straight chain group having 2 to 6 carbon atoms.

A particularly preferred zwitterionic monomer is2-methacryloyloxyethyl-2′-trimethylammonium ethyl phosphate inner salt(MPC).

Another suitable monomer is a compound which is a mono-, di-, or oligo-hydroxy C₂₋₆ alkyl(alk) acrylate or -(alk) acrylamide, an oligo (ethoxy)alkyl(alk)acrylate or acrylamide or a N,N-dimethyl(alk)acrylamide.

Where X² is a polymer chain the reagent of the general formula II may bemade in a preliminary polymerisation step in which ethylenicallyunsaturated monomers are polymerised in the presence of an initiator ofthe general formula XI

in which X³, L² and R¹ to R⁵ are as defined in relation to generalformula II and X⁴ is selected from the group consisting of Cl, Br, I,OR⁵¹, SR⁵², SeR⁵², OP(═O)R⁵², OP(═O)(OR⁵²)₂, O—N(R⁵²)₂ andS—C(═S)N(R⁵²)₂, where R⁵¹ is alkyl of from 1 to 20 carbon atoms in whicheach of the hydrogen atoms may be independently replaced by halide, R⁵²is aryl or a straight or branched C₁-C₂₀ alkyl group, and where anN(R⁵²)₂ group is present, the two R⁵² groups may be joined to form a 5-or 6-membered heterocyclic ring.

In this atom or group transfer radical polymerisation process theethylenically unsaturated monomers which are polymerised may be of thesame type as are described above in relation to polymerisation processesin which the amide conjugate is used as the initiator.

A group or atom transfer radical polymerisation is carried out in thepresence of a catalyst, which comprises a transition metal salt and aligand. The transition metal compound which comprises a component of thecatalyst is M_(q) ^(n+)X⁵ _(q), where:

M_(t) ^(q+) may be selected from the group consisting of Cu¹⁺, Cu²⁺,Fe²⁺, Fe³⁺, Ru²⁺, Ru³⁺, Cr²⁺, Cr³⁺, Mo²⁺, Mo³⁺, W²⁺, W³⁺, Mn²⁺, Mn³⁺,Mn⁴⁺, Rh³⁺, Rh⁴⁺, Re²⁺, Re³⁺, Co⁺, Co²⁺, Co³⁺, V²⁺, V³⁺, Zn⁺, Zn²⁺,Ni²⁺, Ni³⁺, Au⁺, Au²⁺, Ag⁺ and Ag²⁺;

X⁵ is selected from the group consisting of halogen, C₁-C₆-alkoxy,(SO₄)_(1/2), (PO₄)_(1/3), (R¹⁸PO₄)½ (R¹⁸ ₂PO₄), triflate,hexafluorophosphate, methanesulphonate, arylsulphonate, CN and R¹⁹CO₂,where R¹⁸ is aryl or a straight or branched C₁₋₂₀ alkyl and R¹⁹ is H ora straight or branched C₁-C₆ alkyl group which may be substituted from 1to 5 times with a halogen; and

q is the formal charge on the metal (0≦n≦7).

Preferably X⁵ is halide, most preferably chloride or bromide.Particularly suitable transition metal compounds are based on copper orruthenium, for instance CuCl or RuCl₂.

In the catalyst, the ligand is preferably selected from the groupconsisting of:

a) compounds of the formulas:R³⁷-Z-R³⁸ andR³⁷-Z-(R³⁹-Z)_(p)-R³⁸where:

R³⁷ and R³⁸ are independently selected from the group consisting of H,C₁-C₂₀ alkyl, aryl, heterocyclyl and C₁-C₆ alkoxy, C₁-C₄ dialkylamino,C(═O)R⁴⁰, C(═O)R⁴¹R⁴² and A⁸C(═O)R⁴³, where A⁸ may be NR⁴⁴ or O; R⁴⁰ isalkyl of from 1 to 20 carbon atoms, aryloxy or heterocyclyloxy; R⁴¹ andR⁴² are independently H or alkyl of from 1 to 20 carbon atoms or R⁴¹ andR⁴² may be joined together to form an alkanediyl group of from 2 to 5carbon atoms, thus forming a 3- to 6-membered ring; R⁴³ is H, straightor branched C₁-C₂₀ alkyl or aryl and R⁴⁴ is hydrogen, straight orbranched C₁₋₂₀-alkyl or aryl; or R³⁷ and R³⁸ may be joined to form,together with Z, a saturated or unsaturated ring;

Z is O, S, NR⁴⁵ or PR⁴⁶, where R⁴⁶ is selected from the same group asR³⁷ and R³⁸, and where Z is PR⁴⁶, R⁴⁶ can also C₁-C₂₀ alkoxy or Z may bea bond, CH₂ or a fused ring, where one or both of R³⁷ and R³⁸ isheterocyclyl,

each R³⁹ is independently a divalent group selected from the groupconsisting of C₁-C₈ cycloalkanediyl, C₁-C₈ cycloalkanediyl, arenediyland heterocyclylene where the covalent bonds to each Z are at vicinalpositions or R³⁹ may be joined to one or both of R³⁷ and R³⁸ toformulate a heterocyclic ring system; and

p is from 1 to 6;

b) CO;

c) porphyrins and porphycenes, which may be substituted with from 1 to 6halogen atoms, C₁-₆ alkyl groups, C₁₋₆-alkoxy groups, C₁₋₆alkoxycarbonyl, aryl groups, heterocyclyl groups, and C₁₋₆alkyl groupsfurther substituted with from 1 to 3 halogens;

d) compounds of the formula R⁴⁷ R⁴⁸C(C(═O)R⁴⁹)₂, where R⁴⁹ isC₁₋₂₀alkyl, C₁₋₂₀ alkoxy, aryloxy or heterocyclyloxy; and each of R⁴⁷and R⁴⁸ is independently selected from the group consisting of H,halogen, C₁₋₂₀ alkyl, aryl and heterocyclyl, and R⁴⁷ and R⁴⁸ may bejoined to form a C₁₋₈ cycloalkyl ring or a hydrogenated aromatic orheterocyclic ring, of which the ring atoms may be further substitutedwith 1 to 5 C₁₋₆alkyl groups, C₁₋₆alkoxy groups, halogen atoms, arylgroups, or combinations thereof; and

e) arenes and cyclopentadienyl ligands, where said cyclopentadienylligand may be substituted with from one to five methyl groups, or may belinked through and ethylene or propylene chain to a secondcyclopentadienyl ligand.

Selection of a suitable ligand is, for instance, based upon thesolubility characteristics and/or the separability of the catalyst fromthe product polymer mixture. Generally the catalyst is soluble in theliquid reaction mixture, although under some circumstances it may bepossible to immobilise the catalyst, for instance on a porous substrate.For the preferred process, which is carried out in the liquid phase, theligand is soluble in a liquid phase. The ligand is generally a nitrogencontaining ligand. The preferred ligand may be a compound including apyridyl group and an imino moiety, such as bipyridine, or

where R⁵⁰ is a suitable alkyl group, the substituent being variable andadaptable to confer desired solubility characteristics or may betriphenylphosphine or 1,1,4,7,10,10-hexamethyl-triethylene tetramine.

Such nitrogen-containing ligands are usefully used in combination withcopper (I) chloride, copper (I) bromide or ruthenium chloride transitionmetal compounds as part of the catalyst.

The atom or group transfer radical polymerisation process of theinvention is preferably carried out to achieve a degree ofpolymerisation in the range 2 to 100. Preferably the degree ofpolymerisation is in the range 5 to 50, more preferably in the range 10to 25. In the preferred group or atom transfer radical polymerisationtechnique, the degree of polymerisation is directly related to theinitial ratios of initiator to monomer. Preferably the ratio is in therange 1:(2 to 100), more preferably in the range of 1:(5 to 50), mostpreferably in the range 1:(10 to 25).

The ratio of metal compound and ligand in the catalyst should beapproximately stoichiometric, based on the ratios of the components whenthe metal ion is fully complexed. The ratio should preferably be in therange 1:(0.5 to 2) more preferably in the range 1:(0.8 to 1.25).Preferably the range is about 1:1.

In the process, the catalyst may be used in amounts such that a molarequivalent quantity as compared to the level of initiator is present.However, since catalyst is not consumed in the reaction, it is generallynot essential to include levels of catalyst as high as of initiator. Theratio of catalyst (based on transition metal compound) to initiator ispreferably in the range 1:(1 to 50), more preferably in the range 1:(1to 10).

In the invention R⁵ is, for instance, a group joined to the carbon atomto which R³ and R⁴ are joined through an oxygen atom, a group —NR⁸—where R⁸ is hydrogen or C₁₋₄ alkyl, a carboxyl (provided that R³ and R⁴together are not ═O) or a alkyl carbon atom or aryl carbon atom. It mayadditionally comprise an alkanediyl group, cycloalkane-diyl, anoligo(alkoxy) alkyl, arylene or alkarylene group. At the end joined toL² it may comprise —O—, —S—, —CO— or NR⁸ or may be joined to L² throughan aromatic or aliphatic carbon atom.

Linkers L¹ and/or L² may comprise, for instance, the residue of abifunctional linking reagent such as an oligo peptide, a compound havingtwo similar functionalities, such as isocyanates epoxides hydroxyl,thiols, amines, carboxyls, aldehydes, or two different functionalitiesselected from the same list. Preferably where L¹ and/or L² is other thana bond, it is the residue of a hetero-bifunctional linking reagentespecially a reagent comprising one carboxylic functionality and anamide, hydroxy or phenol functionality.

The invention allows conjugation of an biologically active moiety R to apolymer or initiator precursor of a polymer, using well understoodconjugation reactions, which may be carried out under relatively mildconditions and with high efficiency, to form conjugates havingcontrollable solubility, bioavailability, stability or delivery andtargeting characteristics. For instance protein actives may be renderedmore stable by conjugation to hydrophilic polymers, especially polymershaving pendant zwitterionic groups. The active may, for instance be anantibody or fragment thereof, a cytokine, such as an integer or, apeptide therapeutic, a hormone, an enzyme. The polymer conjugated moietymay be cleavable after delivery, for instance where it is conjugated atan active site, or may be substantially non-cleavable provided theactivity of the biologically active compound is not deleteriouslyaffected. The biologically active moiety may be linked to L¹X¹ throughfunctional pendant groups or terminal groups. On peptides the pendantgroups may be side chains on amino acyl residues, especially amine,hydroxyl thiol or carboxyl groups. Similarly, on such compounds theterminal groups are amino groups or carboxyl groups. Since r may begreater than 1, this indicates that more than one of the potentiallyreactive functional pendant or terminal groups may be provided with L¹X¹groups for subsequent conjugation.

Preliminary steps of attaching linkers and groups X¹ to biologicallyactive molecules, including methods for controlling the site of theattachment, by suitable protection and activation strategies, are knownand may be used herein.

The present invention also provides a conjugate of the general formulaXII

where B¹ is —CONR⁶— or —NR⁶—CO—

t is in the range 1 to r; and

R, L¹, L², R¹ to R⁵ and X² are as defined above, as well as compositionscomprising the compound and the compound for use in a method oftreatment or diagnosis. The value of t may be an integer but need not bean integer as it will represent the average level of derivatisationacross the population. Where t=r the biologically active molecule isfully derivatised.

The compound of formula XII, especially where it contains a linker L¹ orL², may be formed in an alternative sequence of steps in which thebiologically active compound is conjugated to an intermediate reagent bya process other than amide bond formation. For instance the intermediatemay be formed in a preliminary reaction in which the bond B¹ is formed.An example is described below in Example 4. The process of the firstaspect of the invention is the preferred route for synthesising XII.

The invention also provides a polymerisation process in whichethylenically unsaturated monomers are polymerised by atom or groupradical transfer polymerisation in the presence of an initiationselected from 4-(3-(2-bromo, 2-methyl-propionate)phenyl)-propionic acidN-hydroxysuccinimide ester and 2-bromo, 2-methyl-propionic acidN-hydroxysuccinimide ester. The monomers in the polymerisation arepreferably as defined above in relation to the first aspect of theinvention. The polymerisation is conducted in the same preferredsolvents, preferably including water.

The compound 2-bromo, 2-methyl-propionic acid N-hydroxysuccinimide esteris believed to be a new compound and is thus claimed herein.

Polymer—biologically active compound conjugates may be usefulpharmaceuticals eg as the compound may be a prodrug. The conjugation maybe used to control the solubility biological availability, stability,immunogenicity or other physical, chemical or biological characteristicsof the biologically active compound.

The invention is illustrated in the following examples:

in which we describe a method by which the 2-bromo, 2-methyl propionicacid bromide can be converted in the first instance to a water-solubleand reactive analogue that can be subsequently used to modify biologicalentities in aqueous media in order to prepare initiators for ATRP. Theaqueous-reactive initiator can be attached to the biological moleculeand then the entire entity used to initiate ATRP in water, oralternatively, the reactive initiator itself can be used to grow apolymer chain with a reactive functionality, which can be subsequentlyreacted with the biological entity in a later reaction to form thepolymer-modified molecule.

EXAMPLE 1a Preparation of 4-(3-(2-bromo,2-methyl-propionate)phenyl)-propionic acid N-hydroxysuccinimide ester

To a solution of 4-(3-hydroxyphenyl)-propionic acid (a) in acetonitrile,TMEDA (0.55 equiv) is added and stirred at room temperature for about 5min. A solution of 2-bromo, 2-methyl propionic acid bromide (b) (1.5equivalent) in acetonitrile is slowly added. After about 15 min ofaddition a white precipitate should be observed in the reaction vessel.After addition of the acid bromide (about 30 min), the reaction wasstirred for a further 60 min approximately. The reaction mixture isfiltered and the solvent was removed in vacuo to yield 4-(3-(2-bromo,2-methyl-propionic ester)phenyl)-propionic acid (c).

To a solution of 4-(3-(2-bromo, 2-methyl-propionate)phenyl)-propionicacid (1 equivalent) in THF, N-hydroxy succinimde (1.05 equivalents) anddicyclohexylcarbodi-imide (d) (1.05 equivalents) is added at −18° C. andstirred for about 2 h. The reaction was allowed to warm to roomtemperature and stirred for a further 10 h approximately. The reactionis worked up as described by Rutinger & Ruegg in Biochem J., 133(3),538, 1973 to yield 4-(3-(2-bromo, 2-methyl-propionate)phenyl)-propionicacid N-hydroxysuccinimide ester (e).

EXAMPLE 1b Preparation of 2-bromo, 2-methyl-propionic acidN-hydroxysuccinimide ester

To a solution of 2-bromo, 2-methyl-propionic acid (a1) (1 equivalent) inTHF, N-hydroxy succinimde (1.05 equivalents) anddicyclohexylcarbodi-imide (d) (1.05 equivalents) is added at −18° C. andstirred for about 2 h. The reaction is allowed to warm to roomtemperature and stirred for about a further 10 h. The reaction is workedup as described by Rutinger & Ruegg in Biochem J., 133(3), 538, 1973 toyield 2-bromo, 2-methyl-propionic acid N-hydroxysuccinimide ester (e1).

EXAMPLE 2

ATRP Using a Lysozyme-modified Initiator

a) To a suspension of 4-(3-(2-bromo, 2-methyl-propionate)phenyl)-propionic acid N-hydroxysuccinimide (e) ester in borate buffer,lysozyme is added and the resulting mixture gently shaken at roomtemperature for about 8 h. The initiator (f) was used without isolation.

b) The initiator solution is purged with nitrogen for about 30 min, andthen copper bromide catalyst and the bipyridyl ligand added. Thesolution is further purged with nitrogen, and MPC added (50× catalystconcentration, target Mn 15 000). The green reaction mixture was stirredfor about 8 h at room temperature. The reaction is monitored by NMRaliquots, for consumption of the MPC methacrylate groups. The reactionmixture is analysed and product (g) purified by CapillaryElectrophoresis. The conjugated lysozyme may be tested for its activityby a standard assay.

EXAMPLE 3 ATRP Using the4-(3-(2-bromo-2-methyl-propionate)phenyl)-propionic acidN-hydroxysuccinimide ester

a) The initiator solution(4-(3-(2-bromo-2-methyl-propionate)phenyl)-propionic acidN-hydroxysuccinimide ester (e) in methanol) is purged with nitrogen forabout 30 min, and then copper bromide catalyst and the bipyridyl ligandadded. The solution was further purged with nitrogen, and MPC added (50×catalyst concentration, target Mn 15 000). The green reaction mixture isstirred for about 8 h at room temperature. The reaction is monitored byNMR aliquots, for consumption of the MPC methacrylate groups. Thereaction mixture is purified on a silica gel column to yield4-(3-(2-poly-Pm, 2-methyl-propionate)phenyl)-propionic acidN-hydroxysuccinimide ester (h).

b) To a suspension of 4-(3-(2-poly-Pm,2-methyl-propionate)phenyl)-propionic acid N-hydroxysuccinimide ester inborate buffer, lysozyme is added and the resulting mixture stirred atroom temperature for about 12 h. The reaction mixture is again analysedand the product (g) purified by Capillary Electrophoresis. The activityof the conjugated lysozyme may be determined by a standard assay.

EXAMPLE 4 N-butyraldehyde 2-bromoisobutylamide

To 100 ml of dry THF, 8.1 g (0.05 mol) 4-aminobutyraldehyde diethylacetal and 8.4 ml (0.06 mol) triethylamine were added. The reactionflask was kept in ice-water bath, and 2-bromoisobutyryl bromide (7.5 ml,0.06 mol) was added dropwise by a syringe over 30 min. The reaction wasthen stirred for 2 hours at room temperature. The white amine halidesalt precipitate was filtered off by Buchner funnel. After evaporatinghalf of the THF under reduced pressure, the filtered THF solution wastreated with 25 ml of 20% trifluoroacetic acid aqueous solutionovernight to cleave the diethyl acetal protection group. The mixture wasneutralised by addition of 5% of NaHCO₃ water solution to pH 8, and theproduct aldehyde was extracted three times by 50 ml DCM each time. Afterevaporation of DCM, a red-yellow liquid was obtained. Furtherpurification was carried out by silicon column using DCM as eluent. Thefinal product is colorless liquid, and stored at −20° C. freezer.

The product may be conjugated to lysozyme by a reductive aminationprocess in which a Schiff base is formed. This general technique isknown for derivatising proteins having available primary amine groups.

1-43. (canceled)
 44. A process forming a biologically active conjugatecompound comprising conjugation step in which a biologically activestarting material of the general formula I

is reacted in an amide bond forming step with a reagent of the generalformula II

to form an amide-linked conjugate, in which R is a biologically activemoiety; L¹ is a bond or a divalent organic linker; and X¹ is —NHR⁶ or—COR⁷, in which R⁶ is selected from the group consisting of hydrogen,C₁₋₆ alkyl, aryl and amine activating group and R⁷ is hydroxyl or acarboxyl activating group; X² is selected from the group consisting of apolymer formed from ethylenically unsaturated monomers and joinedthrough a terminal group, Cl, Br, I, OR¹⁰, SR¹⁴, SeR¹⁴, OP(═O)R¹⁴,OP(═O)(OR¹⁴)₂, O—N(R¹⁴)₂ and S—C(═S)N(R¹⁴)₂, where R¹⁰ is alkyl of from1 to 20 carbon atoms in which each of the hydrogen atoms may beindependently replaced by halide, R¹⁴ is aryl or a straight or branchedC₁-C₂₀ alkyl group, and where an N(R¹⁴)₂ group is present, the two R¹⁴groups may be joined to form a 5- or 6-membered heterocyclic ring; R¹and R² are each independently selected from the group consisting of H,halogen, C₁-C₂₀ alkyl, C₃-C₈ cycloalkyl, C(═O)R¹⁵, C(═O)NR¹⁶R¹⁷, COCl,OH, CN, C₂-C₂₀ alkenyl, oxiranyl, glycidyl, aryl, heterocyclyl, aralkyland aralkenyl, in any of which the alkyl, alkenyl or aryl, heterocyclylor cycloalkyl groups there may be from 1 to 3 substituents selected fromthe group consisting of hydrogen, hydroxy C₁-C₄ alkoxy, acyloxy, aryl,heterocyclyl, C(═O)R¹⁵, C(═O)NR¹⁶R¹⁷, oxyranyl and glycidyl; R¹⁵ isselected from the group consisting of alkyl of from 1 to 20 carbonatoms, alkoxy of from 1 to 20 carbon atoms, oligo(alkoxy) in which eachalkoxy group has 1 to 3 carbon atoms, aryloxy and heterocyclyloxy, anyof which groups may have substituents selected from the group consistingof optionally substituted alkoxy, oligoalkoxy, amino and hydroxylgroups; R¹⁶ and R¹⁷ are independently selected from the group consistingof H and alkyl of from 1 to 20 carbon atoms which alkyl groups, may havesubstituents selected from the group consisting of alkoxy, acyl,acyloxy, alkoxycarbonyl, alkenoxycarbonyl, aryl and hydroxy, or R¹⁶ andR¹⁷ may be joined together to form an alkanediyl group of from 2 to 5carbon atoms, thus forming a 3- to 6-membered ring; R³ and R⁴ areindependently selected from the group consisting of hydrogen, and C₁₋₆alkyl, or R³ and R⁴ together are ═O or ═NR⁸ where R⁸ is hydrogen or C₁₋₄alkyl; R⁵ is selected from the group consisting of a bond, —O—, —S— anddivalent organic groups; L² is a bond or a divalent linker and, where X¹is NHR⁶, X³ is COR⁷ and where X¹ is COR⁷, X³ is NHR⁶ and r is an integerof at least
 1. 45. A process according to claim 44 in which X¹ is NHR⁶and X³ is COR⁷.
 46. A process according to claim 45 in which R⁶ isselected from the group consisting of C₁₋₆ alkyl, aryl and H.
 47. Aprocess according to claim 44 in which R⁷ is N-succinimidyloxy.
 48. Aprocess according to claim 44 in which the compound of formula I is aprotein.
 49. A process according to claim 44 in which the amide bondforming step is carried out in a solvent system comprising a proticsolvent.
 50. A process according to claim 47 in which X² is selectedfrom the group consisting of Cl, Br, I, OR¹⁰, SR¹⁴, SeR¹⁴, OP(═O)R¹⁴,OP(═O)(OR¹⁴)₂, O—N(R¹⁴)₂ and S—C(═S)N(R¹⁴)₂, where R¹⁰ is alkyl of from1 to 20 carbon atoms in which each of the hydrogen atoms may beindependently replaced by halide, R¹⁴ is aryl or a straight or branchedC₁-C₂₀ alkyl group, and where an N(R¹⁴)₂ group is present, the two R¹⁴groups may be joined to form a 5- or 6-membered heterocyclic ring.
 51. Aprocess according to claim 50 in which the product of the amide bondforming is used as the initiator in an atom or group transfer radicalpolymerisation step of ethylenically unsaturated monomers.
 52. A processaccording to claim 44 in which X² is a polymer chain.
 53. A processaccording to claim 52 in which the compound of formula II is made in apreliminary polymerisation step in which ethylenically unsaturatedmonomers are polymerised in the presence of an initiator of the generalformula XI

in which X³, L² and R¹ to R⁵ are as defined in claim 1 in relation togeneral formula II and X⁴ is selected from the group consisting of Cl,Br, I, OR⁵¹, SR⁵², SeR⁵², OP(═O)R⁵², OP(═O)(OR⁵²)₂, O—N(R⁵²)₂ andS—C(═S)N(R⁵²)₂, where R⁵¹ is alkyl of from 1 to 20 carbon atoms in whicheach of the hydrogen atoms may be independently replaced by halide, R⁵²is aryl or a straight or branched C₁-C₂₀ alkyl group, and where anN(R⁵²)₂ group is present, the two R⁵² groups may be joined to form a 5-or 6-membered heterocyclic ring.
 54. A process according to claim 53 inwhich the ethylenically unsaturated monomers are water-soluble.
 55. Aprocess according to claim 54 in which the polymerisation step iscarried out in the presence of water.
 56. A process according to claim53 in which the ethylenically unsaturated monomers have the generalformula III

in which R²³ is selected from the group consisting of hydrogen, halogen,C₁₋₄ alkyl and groups COOR²⁷ in which R²⁷ is hydrogen and C₁₋₄ alkyl;R²⁴ is selected from the group consisting of hydrogen, halogen and C₁₋₄alkyl; R²⁵ is selected from the group consisting of hydrogen, halogen,C₁₋₄ alkyl and groups COOR²⁷ provided that R²³ and R²⁵ are not bothCOOR²⁷; and R²⁶ is selected from the group consisting of C₁₋₁₀ alkyl,C₁₋₂₀ alkoxycarbonyl, mono- and di-(C₁₋₂₀ alkyl) amino carbonyl, C₆₋₂₀aryl, C₇₋₂₀ aralkyl, C₆₋₂₀ aryloxycarbonyl, C₇₋₂₀ -aralkyloxycarbonyl,C₆₋₂₀ arylamino carbonyl, C₇₋₂₀ aralkyl-amino carbonyl, hydroxyl andC₂₋₁₀ acyloxy groups, any of which may have one or more substituentsselected from the group consisting of halogen atoms, alkoxy,oligo-alkoxy, aryloxy, acyloxy, acylamino, amine carboxyl, sulphonyl,phosphoryl, phosphino, zwitterionic groups, hydroxyl, vinyloxycarbonyland other vinylic and allylic groups, and reactive silyl and silyloxygroups; or R²⁶ and R²⁵ or R²⁵ and R²³ may together form —CONR²⁸CO inwhich R²⁸ is a C₁₋₂₀ alkyl group.
 57. A process according to claim 56 inwhich R²³ and R²⁴ are hydrogen atoms.
 58. A process according to claim57 in which R²⁶ is selected from groups consisting of alkoxy-carbonyl,(alkyl)amino carbonyl, aryloxycarbonyl and carboxyl group, and R²⁵ ishydrogen or C₁₋₄ alkyl.
 59. A process according to claim 53 in which theethylenically unsaturated monomers include a zwitterionic monomer havingthe general formula IVYBX   IV in which Y is an ethylenically unsaturated group selected fromH₂C═CR¹⁷—CO-A-, H₂C═CR¹⁷—C₆H₄-A¹-, H₂C═CR¹⁷—CH₂A¹⁸,R²O—CO—CR¹⁷═CR¹⁷—CO—O, R¹⁷CH═CH—CO—O—, R¹⁷ CH═C(COOR¹⁸)CH₂—CO—O,

A is —O— or NR¹⁹; A¹ is selected from the group consisting of a bond,(CH₂)_(l)A² and (CH₂)_(l)SO₃— in which l is 1 to 12; A² is selected fromthe group consisting of a bond, —O—, O—CO—, CO—O, CO—NR¹—, —NR¹—CO,O—CO—NR¹—, and NR¹—CO—O—; R¹⁷ is hydrogen or C₁₋₄ alkyl; R¹⁹ ishydrogen, C₁₋₄ alkyl or BX; R¹⁸ is hydrogen or C₁₋₄ alkyl; B is selectedfrom group consisting of a bond, straight and branched alkanediyl,alkylene oxaalkylene, and alkylene (oligooxalkylene) groups, optionallycontaining one or more fluorine substituents; and X is a zwitterionicgroup.
 60. A process according to claim 59 in which X is a group of thegeneral formula V

in which the moieties A³ and A⁴, which are the same or different, areselected from the groups consisting of —O—, —S—, —NH— and a valencebond, and W⁺ is selected from the groups consisting of —W¹—N⁺R²⁰ ₃,—W¹—P⁺R²¹ ₃, —W¹—S⁺R²¹ ₂ and —W¹-Het⁺ in which: W¹ is selected from thegroups consisting of alkanediyl of 1 to 6 carbon atoms optionallycontaining one or more ethylenically unsaturated double or triple bonds,arylene, alkylene arylene, arylene alkylene, alkylene aryl alkylene,cycloalkanediyl, alkylene cycloalkyl, cycloalkyl alkylene and alkylenecycloalkyl alkylene, which group W¹ optionally contains one or morefluorine substituents and/or one or more functional substituent groups;and either the groups R²⁰ are the same or different and each is selectedfrom the groups consisting of hydrogen, alkyl of 1 to 4 carbon atoms andaryl, or two of the groups R²⁰ together with the heteroatom atom towhich they both are attached form an aliphatic heterocyclic ringcontaining from 5 to 7 atoms, or the three groups R²⁰ together with thenitrogen atom to which they are attached as heteroaromatic ring having 5to 7 atoms, either of which rings may be fused with another saturated orunsaturated ring to form a fused ring structure containing from 5 to 7atoms in each ring, and optionally one or more of the groups R²⁰ issubstituted by a hydrophilic functional group, and the groups R²¹ arethe same or different and each is R²⁰ or a group OR²⁰, where R²⁰ is asdefined above; or Het is an aromatic nitrogen-, phosphorus- or sulphur-,containing ring.
 61. A process according claim 59 in which X has thegeneral formula VI

where the groups R²² are the same or different and each is hydrogen orC₁₋₄ alkyl, and m is from 1 to
 4. 62. A process according to claim 59 inwhich Y is H₂C═CR¹⁷CO-A-, in which A is O and R¹⁷ is H or CH₃.
 63. Aprocess according to claim 59 in which B is a straight chainC₂₋₆-alkanediyl group.
 64. A process according to claim 53 in which theethylenically unsaturated monomers include a compound selected from thegroups consisting of mono-, di-, and oligo- hydroxy C₂₋₆ alkyl(alk)acrylates and -(alk) acrylamides, oligo (ethoxy) alkyl(alk)acrylates,acrylamide and N,N-dimethyl(alk)acrylamides.
 65. A process according toclaim 53 which is carried out in the presence of a catalyst whichcomprises a transition metal salt and a ligand.
 66. A process accordingto claim 65 in which the ligand is bipyridine, or

where R⁵⁰ is a suitable alkyl group triphenylphosphine or1,1,4,7,10,10-hexamethyl-triethylene tetramine.
 67. A process accordingto claim 65 in which the transition metal salt is copper (1) chloride,copper (1) bromide or ruthenium chloride.
 68. A process according toclaim 44 in which L¹ and L² are each a bond or an oligopeptidyl group.69. An amide-linked conjugate compound of the general formula XII

where B¹ is —CONR⁶— or —NR⁶—CO—; t is in the range 1 to r; r is aninteger of more than 1; R is a biologically active moiety; L¹ is a bondor a divalent organic linker; X² is selected from the group consistingof a polymer formed from ethylenically unsaturated monomers and joinedthrough a terminal group, Cl, Br, I, OR¹⁰, SR¹⁴, SeR¹⁴, OP(═O)R¹⁴,OP(═O)(OR¹⁴)₂, O—N(R¹⁴)₂ and S—C(═S)N(R¹⁴)₂, where R¹⁰ is alkyl of from1 to 20 carbon atoms in which each of the hydrogen atoms may beindependently replaced by halide, R¹⁴ is aryl or a straight or branchedC₁-C₂₀ alkyl group, and where an N(R¹⁴)₂ group is present, the two R¹⁴groups may be joined to form a 5- or 6-membered heterocyclic ring; R¹and R² are each independently selected from the group consisting of H,halogen, C₁-C₂₀ alkyl, C₃-C₈ cycloalkyl, C(═O)R¹⁵, C(═O)NR¹⁶R¹⁷, COCl,OH, CN, C₂-C₂₀ alkenyl, oxiranyl, glycidyl, aryl, heterocyclyl, aralkyland aralkenyl, in any of which the alkyl, alkenyl or aryl, heterocyclylor cycloalkyl groups there may be from 1 to 3 substituents selected fromthe group consisting of hydrogen, hydroxy, C₁-C₄ alkoxy, acyloxy, aryl,heterocyclyl, C(═O)R¹⁵, C(═O)NR¹⁶R¹⁷, oxyranyl and glycidyl; R¹⁵ isselected from the group consisting of alkyl of from 1 to 20 carbonatoms, alkoxy of from 1 to 20 carbon atoms, oligo(alkoxy) in which eachalkoxy group has 1 to 3 carbon atoms, aryloxy and heterocyclyloxy, anyof which groups may have substituents selected from the group consistingof optionally substituted alkoxy, oligoalkoxy, amino and hydroxylgroups; R¹⁶ and R¹⁷ are independently H or alkyl of from 1 to 20 carbonatoms which alkyl groups, may have substituents selected from the groupconsisting of alkoxy, acyl, acyloxy, alkoxycarbonyl, alkenoxycarbonyl,aryl and hydroxy, or R¹⁶ and R¹⁷ may be joined together to form analkanediyl group of from 2 to 5 carbon atoms, thus forming a 3- to6-membered ring; R³ and R⁴ are independently selected from the groupconsisting of hydrogen and C₁₋₆ alkyl, or R³ and R⁴ together are ═O or═NR⁸ where R⁸ is hydrogen or C₁₋₄ alkyl; R⁵is selected from the groupsconsisting of a bond, —O—, —S— and divalent organic groups; and L² is abond or a divalent linker.
 70. A compound according to claim 69 in whichB¹ is —NR⁶CO— in which R⁶ is H or C₁₋₄ alkyl.
 71. A compound accordingto claim 69 in which X² is a polymer formed from ethylenicallyunsaturated monomers.
 72. A compound according to claim 71 in which theethylenically unsaturated monomers have the general formula III

in which R²³ is selected from the group consisting of hydrogen, halogen,C₁₋₄ alkyl and groups COOR²⁷ in which R²⁷ is hydrogen and C₁₋₄ alkyl;R²⁴ is selected from the group consisting of hydrogen, halogen and C₁₋₄alkyl; R²⁵ is selected from the group consisting of hydrogen, halogen,C₁₋₄ alkyl and groups COOR²⁷ provided that R²³ and R²⁵ are not bothCOOR²⁷; and R²⁶ is selected from the group consisting of C₁₋₁₀ alkyl,C₁₋₂₀ alkoxycarbonyl, mono- and di-(C₁₋₂₀ alkyl) amino carbonyl, C₆₋₂₀aryl, C₇₋₂₀, aralkyl, C₆₋₂₀ aryloxycarbonyl, C₇₋₂₀ -aralkyloxycarbonyl,C₆₋₂₀ arylamino carbonyl, C₇₋₂₀ aralkyl-amino carbonyl, hydroxyl andC₂₋₁₀ acyloxy groups, any of which may have one or more substituentsselected from the group consisting of halogen atoms, alkoxy,oligo-alkoxy, aryloxy, acyloxy, acylamino, amine carboxyl, sulphonyl,phosphoryl, phosphino, zwitterionic groups, hydroxyl, vinyloxycarbonyland other vinylic and allylic groups, and reactive silyl and silyloxygroups; or R²⁶ and R²⁵ or R²⁵ and R²³ may together form —CONR²⁸CO inwhich R²⁸ is a C₁₋₂₀ alkyl group.
 73. A compound according to claim 72in which the ethylenically unsaturated monomers include a zwitterionicmonomer having the general formula IVYBX   IV in which Y is an ethylenically unsaturated group selected fromH₂C═CR¹⁷—CO-A-, H₂C═CR¹⁷—C₆H₄-A¹-, H₂C═CR¹⁷—CH₂A¹⁸,R²O—CO—CR¹⁷═CR¹⁷—CO—O, R¹⁷CH═CH—CO—O—, R¹⁷CH═C(COOR¹⁸)CH₂—CO—O,

A is —O— or NR¹⁹; A¹ is selected from a bond, (CH₂)_(l)A² and(CH₂)_(l)SO₃— in which l is 1 to 12; A² is selected from a bond, —O—,O—CO—, CO—O, CO—NR¹—, —NR¹—CO, O—CO—NR¹—, NR¹—CO—O—; R¹⁷ is hydrogen orC₁₋₄ alkyl; R¹⁹ is hydrogen, C₁₋₄ alkyl or BX; R¹⁸ is hydrogen or C₁₋₄alkyl; B is selected from the group consisting of a bond, straight andbranched alkanediyl, alkylene oxaalkylene, and alkylene(oligooxalkylene) groups, optionally containing one or more fluorinesubstituents; and X is a zwitterionic group.
 74. A compound according toclaim 73 in which X is a group of the general formula V

in which the moieties A³ and A⁴, which are the same or different, areselected from the group consisting of —O—, —S—, —NH— and a valence bond,and W⁺ is selected from the group consisting of —W¹—N⁺R²⁰ ₃, —W¹—P⁺R²¹₃, —W¹—S⁺R²¹ ₂ and —W¹-Het⁺ in which; W¹ is selected from the groupconsisting of alkanediyl of 1-6 carbon atoms optionally containing oneor more ethylenically unsaturated double or triple bonds,disubstituted-aryl (arylene), alkylene arylene, arylene alkylene,alkylene aryl alkylene, cycloalkanediyl, alkylene cycloalkyl, cycloalkylalkylene and alkylene cycloalkyl alkylene, which group W¹ optionallycontains one or more fluorine substituents and/or one or more functionalgroups; and either the groups R²⁰ are the same or different and each isselected from the group consisting of hydrogen alkyl of 1 to 4 carbonatoms, and aryl, or two of the groups R²⁰ together with the nitrogenatom to which they are attached form an aliphatic heterocyclic ringcontaining from 5 to 7 atoms, or the three groups R²⁰ together with theheteroatom to which they are both attached as heteroaromatic ring having5 to 7 atoms, either of which rings may be fused with another saturatedor unsaturated ring to form a fused ring structure containing from 5 to7 atoms in each ring, and optionally one or more of the groups R²⁰ issubstituted by a hydrophilic functional group, and the groups R²¹ arethe same or different and each is R²⁰ or a group OR²⁰, where R²⁰ is asdefined above; or Het is an aromatic nitrogen-, phosphorus- or sulphur-,containing ring.
 75. A compound according to claim 73 in which X has thegeneral formula VI

where the groups R²² are the same or different and each is hydrogen orC₁₋₄ alkyl, and m is from 1 to
 4. 76. A compound according to claim 73in which Y is H₂C═CR¹⁷COA-, in which A is O and R¹⁷ is H or CH₃.
 77. Acompound according to claim 73 in which B is a straight chainC₂₋₆-alkanediyl group.
 78. A compound according to claim 69 in which L¹and L² are each a bond or an oligopeptidyl group.
 79. A compoundaccording to claim 69 in which R is a protein.
 80. A polymerisationprocess in which ethylenically unsaturated monomers are polymerised byatom or group radical transfer polymerisation in the presence of aninitiator selected from 4-(3-(2-bromo,2-methyl-propionate)phenyl)-propionic acid N-hydroxysuccinimide esterand 2-bromo, 2-methyl-propionic acid N-hydroxysuccinimide ester to forma polymer product having an N-hydroxysuccinimide ester terminal group.81. A polymerisation process according to claim 80 in which theethylenically unsaturated monomers have the general formula III

in which R²³ is selected from the group consisting of hydrogen, halogen,C₁₋₄ alkyl and groups COOR²⁷ in which R²⁷ is hydrogen and C₁₋₄ alkyl;R²⁴ is selected from the group consisting of hydrogen, halogen and C₁₋₄alkyl; R²⁵ is selected from the group consisting of hydrogen, halogen,C₁₋₄ alkyl and groups COOR²⁷ provided that R²³ and R²⁵ are not bothCOOR²⁷; and R²⁶ is selected from the group consisting of C₁₋₁₀ alkyl,C₁₋₂₀ alkoxycarbonyl, mono- and di-(C₁₋₂₀ alkyl) amino carbonyl, C₆₋₂₀aryl, C₇₋₂₀ aralkyl, C₆₋₂₀ aryloxycarbonyl, C₇₋₂₀ -aralkyloxycarbonyl,C₆₋₂₀ arylamino carbonyl, C₇₋₂₀ aralkyl-amino carbonyl, hydroxyl andC₂₋₁₀ acyloxy groups, any of which may have one or more substituentsselected from the group consisting of halogen atoms, alkoxy,oligo-alkoxy, aryloxy, acyloxy, acylamino, amine carboxyl, sulphonyl,phosphoryl, phosphino zwitterionic groups, hydroxyl, vinyloxycarbonyland other vinylic and allylic groups, and reactive silyl and silyloxygroups; or R²⁶ and R²⁵ or R²⁵ and R²³ may together form —CONR²⁸CO inwhich R²⁸ is a C₁₋₂₀ alkyl group.
 82. A process according to claim 80carried out in the presence of water.
 83. A process according to claim80 in which the polymer product is reacted with an amine-groupcontaining protein in an amide-bond forming reaction wherein the saidN-hydroxysuccinimide ester group reacts with the said amine group, toform an amide conjugate.
 84. 2-bromo-2-methyl-propionic acidN-hydroxysuccinimide ester.
 85. A process according to claim 51 in whichthe ethylenically unsaturated monomers have the general formula III

in which R²³ is selected from the group consisting of hydrogen, halogen,C₁₋₄ alkyl and groups COOR²⁷ in which R²⁷ is hydrogen and C₁₋₄ alkyl;R²⁴ is selected from the group consisting of hydrogen, halogen and C₁₋₄alkyl; R²⁵ is selected from the group consisting of hydrogen, halogen,C₁₋₄ alkyl and groups COOR²⁷ provided that R²³ and R²⁵ are not bothCOOR²⁷; and R²⁶ is selected from the group consisting of C₁₋₁₀ alkyl,C₁₋₂₀ alkoxycarbonyl, mono- and di-(C₁₋₂₀ alkyl) amino carbonyl, C₆₋₂₀aryl, C₇₋₂₀ aralkyl, C₆₋₂₀ aryloxycarbonyl, C₇₋₂₀ -aralkyloxycarbonyl,C₆₋₂₀ arylamino carbonyl, C₇₋₂₀ aralkyl-amino carbonyl, hydroxyl andC₂₋₁₀ acyloxy groups, any of which may have one or more substituentsselected from the group consisting of halogen atoms, alkoxy,oligo-alkoxy, aryloxy, acyloxy, acylamino, amine carboxyl, sulphonyl,phosphoryl, phosphino, zwitterionic groups, hydroxyl, vinyloxycarbonyland other vinylic and allylic groups, and reactive silyl and silyloxygroups; or R²⁶ and R²⁵ or R²⁵ and R²³ may together form —CONR²⁸CO inwhich R²⁸ is a C₁₋₂₀ alkyl group.
 86. A process according to claim 85 inwhich R²³ and R²⁴ are hydrogen atoms.
 87. A process according to claim86 in which R²⁶ is selected from the group consisting ofalkoxy-carbonyl, (alkyl)amino carbonyl, aryloxycarbonyl and carboxylgroup, and R²⁵ is hydrogen or C₁₋₄ alkyl.
 88. A process according toclaim 51 in which the ethylenically unsaturated monomers include azwitterionic monomer having the general formula IVYBX   IV in which Y is an ethylenically unsaturated group selected fromH₂C═CR¹⁷—CO-A-, H₂C═CR¹⁷—C₆H₄-A¹-, H₂C═CR¹⁷—CH₂A¹⁸, R²O—CO—CR¹⁷—50CR¹⁷—CO—O, R¹⁷CH═CH—CO—O—, R¹⁷CH═C(COOR¹⁸)CH₂—CO—O,

A is —O— or NR¹⁹; A¹ is selected from the group consisting of a bond,(CH₂)_(l)A² and (CH₂)_(l)SO₃— in which l is 1 to 12; A² is selected fromthe group consisting of a bond, —O—, O—CO—, CO—O, CO—NR¹—, —NR¹—CO,O—CO—NR¹—, and NR¹—CO—O—; R¹⁷ is hydrogen or C₁₋₄ alkyl; R¹⁹ ishydrogen, C₁₋₄ alkyl or BX; R¹⁸ is hydrogen or C₁₋₄ alkyl; B is selectedfrom group consisting of a bond, straight and branched alkanediyl,alkylene oxaalkylene, and alkylene (oligooxalkylene) groups, optionallycontaining one or more fluorine substituents; and X is a zwitterionicgroup.
 89. A process according to claim 88 in which X is a group of thegeneral formula V

in which the moieties A³ and A⁴, which are the same or different, areselected from the group consisting of —O—, —S—, —NH— and a valence bond,and W⁺ is selected from the group consisting of —W¹—N⁺R²⁰ ₃, —W¹—P⁺R²¹₃, —W¹—S⁺R²¹ ₂ and —W¹-Het⁺ in which: W¹ is selected from the groupsconsisting of alkanediyl of 1 to 6 carbon atoms optionally containingone or more ethylenically unsaturated double or triple bonds, arylene,alkylene arylene, arylene alkylene, alkylene aryl alkylene,cycloalkanediyl, alkylene cycloalkyl, cycloalkyl alkylene and alkylenecycloalkyl alkylene, which group W¹ optionally contains one or morefluorine substituents and/or one or more functional substituent groups;and either the groups R²⁰ are the same or different and each is selectedfrom the groups consisting of hydrogen, alkyl of 1 to 4 carbon atoms andaryl, or two of the groups R²⁰ together with the heteroatom atom towhich they both are attached form an aliphatic heterocyclic ringcontaining from 5 to 7 atoms, or the three groups R²⁰ together with thenitrogen atom to which they are attached as heteroaromatic ring having 5to 7 atoms, either of which rings may be fused with another saturated orunsaturated ring to form a fused ring structure containing from 5 to 7atoms in each ring, and optionally one or more of the groups R²⁰ issubstituted by a hydrophilic functional group, and the groups R²¹ arethe same or different and each is R²⁰ or a group OR²⁰, where R²⁰ is asdefined above; or Het is an aromatic nitrogen-, phosphorus- or sulphur-,containing ring.
 90. A process according to claim 88 in which X has thegeneral formula VI

where the groups R²² are the same or different and each is hydrogen orC₁₋₄ alkyl, and m is from 1 to
 4. 91. A process according to claim 88 inwhich Y is H₂C═CR¹⁷CO-A-, in which A is O and R¹⁷ is H or CH₃.
 92. Aprocess according to claim 88 in which B is a straight chainC₂₋₆-alkanediyl group.
 93. A process according to claim 51 in which theethylenically unsaturated monomers include a compound selected from thegroups consisting of mono-, di-, are oligo- hydroxy C₂₋₆ alkyl(alk)acrylates and -(alk) acrylamides, oligo (ethoxy) alkyl(alk)acrylates,acrylamide and N,N-dimethyl(alk)acrylamides.
 94. A process according toclaim 51 which is carried out in the presence of a catalyst whichcomprises a transition metal salt and a ligand.
 95. A process accordingto claim 94 in which the ligand is bipyridine, or

where R⁵⁰ is a suitable alkyl group triphenylphosphine or1,1,4,7,10,10-hexamethyl-triethylene tetramine.
 96. A process accordingto claim 94 in which the transition metal salt is copper (1) chloride,copper (1) bromide or ruthenium chloride.
 97. A composition comprising acompound according to claim 69 and an excipient.
 98. A compositioncomprising a compound according to claim 71 and an excipient.
 99. Acomposition comprising a compound according to claim 79 and anexcipient.
 100. A compound according to claim 71 in which R is aprotein.